Werner syndrome (WS) is a hereditary premature aging disorder characterized by chromosomal instability. The WRN gene product is a helicase/exonuclease that presumably functions in DNA metabolism to preserve genome integrity. To understand the DNA structures and cellular pathways that WRN impacts, we have systematically examined the DNA substrate preferences of WRN helicase for unwinding and its interactions with human nuclear proteins. Our biochemical studies indicate that WRN preferentially unwinds DNA replication structures in a defined orientation and utilizes specific DNA structures for recognition. We have initiated a kinetic analysis of WRN helicase activity to define the mechanism of DNA unwinding. To further understand its molecular functions, we have characterized the functional interaction of WRN protein with human Flap Endonuclease 1 (FEN-1), a structure-specific nuclease implicated in DNA repair, replication, and recombination. WRN and FEN-1 form a complex in vivo that co-localizes with arrested replication forks. To better understand the mechanism of action by the WRN-FEN-1 complex, we have investigated their organized function to process branch-migrating DNA structures associated with the replication fork. These studies reveal helicase-dependent and helicase-independent mechanisms for coordinate nucleolytic processing that is DNA structure-specific. It has been proposed that the helicase-endonuclease Dna2 and Rad27 (FEN-1) act sequentially to remove long 5? flaps during Okazaki fragment maturation in S. cerevisiae. Yeast genetic complementation analysis of dna2 mutants was used to further examine the biological significance of the WRN-FEN-1 interaction. Our results indicate that ectopic expression of human WRN in a dna2-1 mutant background rescues the replication and repair phenotypes. Genetic complementation of dna2-1 does not require WRN catalytic activity since expression of a WRN protein fragment devoid of enzymatic activity complements the mutant. We suggest that RecQ helicases modulate Rad2 nucleolytic processing of key DNA replication and repair intermediates to preserve genomic integrity.